TiPS -August

1992 [Vol. 231

299

New mutants to explore nicotinic receptor functions Pharmacological receptors linked to ion channels are entirely membrane-bound and are specialized in chemc+electrical transduction. Their sophisticated architecture reflects their function. The nicotinic acetylcholine receptors from Torpedo electric organ and skeletal muscle are composed of five subunits tightly assembled into a transmembrane pentameric oligomer ar$yS (reviewed in Refs l-3); but in adult muscles of some species (cow, rat, mouse) an l subunit replaces the y subunit. In neural tissues, two main types of subunits have so far been identified that include seven distinct 1y and four different B subunits2. Recent mutagenesis experiments on these nicotinic receptors have revealed novel information on their functional organization. Assembly of subunits Recombinant DNA technology has only recently been used to analyse how the subunits assemble into a defined oligomer that behaves as an acetylcholine-gated ion channel. First, analysis of the diverse channel properties of combinations of wild-type and mutated cDNAs, encoding the brain 014and 82 subunits, demonstrated that a functional oligomer is a pentamer composed of two (Y and three B subunits (i.e. ar2Bs)“. Secondly, peptide elements involved in the assembly of the subunits into a functional oligomer have been identified. Introduction of new consensus sequences for glycosylation (or replacing existing ones) confirmed that the N-terminal domain up to residue 207 of the mouse muscle (Y subunit is translocated across the endoplasmic reticulum membrane, and thus faces the synaptic clefts. Furthermore, using chimeric subunit cDNAs, Hall and co-worker&’ confirmed the transmembrane arrangement13. They showed that there are four membrane-spanning segments, the C-terminus being outside’, and demonstrated that the recognition between sub-

units takes place primarily at the level of the extracellular Nterminal domain6*9. Thus, despite their critical function in ion translocation, the hydrophobic uncharged segments seem to play little part in the specificity of interaction between subunits. L&and-binding site mutated again Nicotinic receptor agonists cause the opening of the ion channel when they interact with two pharmacologically distinct binding sites per receptor oligomer. Affinity labelling experiments with both the reduced and native receptor have shown that these sites are primarily carried by the a: subunits and are plausibly located at their interface with the non-o subunits (reviewed in Refs 10-K?). A major insight into the structure of these sites was provided by affinity and photoaffinity labelling experimentsi3-19. In particular, a significant improvement in the labelling method was achieved by photoselection of the bound ligand (the competitive antagonist DDF) using an energy transfer process. DDF led to the identification of Tyrl90, Cys192, Cys193 and Tyr198 (on the Torpedo IX subunit) as contributing to the ligand-binding area of the receptor (Fig. l), since labelling by DDF was blocked by carbamylcholine and snake cW-toxind5J8. These data, like those subsequently obtained with lophotoxin16 and nicotinel’, confirmed the initial observation made with MBTA13,14. The stretch of amino acids surrounding Cys192 and Cys193 of the Torpedo receptor contributes to the binding of acetylcholine and snake o-toxins (Fig. 1). The results also suggested the participation of several Tyr residues within this stretchis-l*. A novel finding was that two additional peptide loops, one including Trp86 and Tyr93 (which is also labelled by acetylcholine mustard”) and the other Trp149 and Tyrl51, are accessible to the

bound ligand15r’s. It was also demonstrated that the extent of iabelling of these two additional loops increases if the conformation of the receptor protein changes from low affinity (‘resting’) to high affinity (‘desensitized’)iO. Mutagenesis experiments have now shown that the labelled amino acids from these three peptide loops do contribute to the pharmacological response of the receptor to agonists and snake (Ytoxins. Early mutagenesis experiments in Torpedo or mouse muscle receptor, replacing Cys192 or Cys193 by Ser (Ref. 20), or Tyrl90 by Phe (Ref. 21) (Fig. l), resulted in a lo- to 50-fold decrease of the apparent affinity for acetylcholine and a reduction of cw-toxin binding. In recent experiments, mutations placed Phe residues into the sites of residues homologous to Tyr93, Trp149 or Tyrl90 in the neuronal homoligomeric o7 receptor (in single-letter code, the mutants Y92F, Wl48F and Yl87F, respectively). These mutations decreased the apparent affinity of the binding site for acetylcholine (lo- to lOO-fold) and nicotine (350fold in the case of W148F), and were also shown to affect 01bungarotoxin bindinp (Fig. 1). Yet, replacements of these aromatic amino acids by Ser residues completely abolished the ionic response. Crosslinking and mutagenesis experiments together stress the functional relevance of aromatic amino acids from these distinct loops of the large N-terminal domain to the complexation of the quaternary ammonium group of the ligands. Interestingly, aromatic amino acids have been found in the ligand-binding sites of anti-phosphorylcholine antibodies and of acetylcholinesterase= as in three-dimensional models of the muscarinic acetylcholine receptor. Also, sydWtiC macrocycles made of strictly a~+ matic rings display up to 50 w affinity for acetylcholine (see Refs 22 and 24 and refs therein). The presence of aromatic sidechains in the binding sites for quaternary ammonium ligands may thus be a property shared by several acetylcholine-binding proteins. Other amino acid sidechains may also play a critical role in receptor function. Indeed, mutation of the highly conserved @19X.El~vierkien~PublishcnLtd(UK)

rensptor

tqg. t. t_o&km m ti Torpedo a; fi y and 6 nicotinic acetylcholine subunitsof amino acids ider!tified by aWin9 /abelling and siteam as ~ytng a et d in tigand binding or ion transtooation.Above: amino ao!ds that conmbute to the nicotinic . . a@witha,rheQsubw~itarebundin tbree disttftctdomains by [abettingwith DDF(*), acelycholine mustard(m). fophotoxin ~~ (r). Q&Q a& Cyst&3 were fnit&#y labetfed by MBTA (01. stain of the ~~~,of ?I?ii?o aCi$s Tyr93. Trpf!9 d TF~JK~ & phe in ~r7 newmat and mu&e cfyrlso) receptor decreases the -rent ahimty for fficotmIc agomsts as do srmdar e of cy~f@ & @sts3. A.&at&n (+) of Aspzoo convertspet&# agonists into competitive an&onists and may be involved in w-s mm: amino a&& (0) /a&&/edby the channel blocker chlorpromaztne define three rings referred to as the Thr, sar w t.w &gs within the h&? ttansmembrane doma!n. The Ser ring is also Welled by trtphenylmethytphosphonium. Mutations (+) in tfwras three rings cause &anges of propertfes of the ion channel, as in the cytopfasmic, iniermediaie and oUagr charged rings. Mqroadifen mustard labefs the outer rfng andrmqacdhe for ~~~~e~ a2ikfelabefs the end of M1. abbe in the t&u ting strikingly modify desensitizatfon and a# assumed to a/tow ion conductance in the desensitized state. Datafrom Refs 13-22,25-35,37,39,43 and 44.

LIPSTSSAVP L@AVTVPLLLL

VPETSLSVP KVPETSLNVP

LLAQAVFLL t

RLPETALAVP

t

mepacrine azide

Asp200 residue in the ToTedo ar subunit converts partial agonists (phenyltrimethyl ammonium or tetramethyl ammonium) of the wild-type receptor into competitlve a~~tagonists~, without significantly decreasing the apparent affinity for acetylcholine. The D2OON mutation thus seems to interfere with the coupling between agonist binding and channel opening, and/or to alter the difference between the phannacological properties of the acetylcholine receptor in the resting and active confo~atio~s, Mapping by mutagenesis Photolabelllng the ion channel with channel blockers such as chlorpromazine (as in Fig. 1) or ~pheny~ethyl phosphonium first pointed to the hydrophobic transmembrane segment M2 being a component of the ion chan-

chlorpromazine

nel*@4 Site-directed mutagenesis experiments soon corroborated these observations31J7, resulting in the distinction of two major categories of amino acid rings: uncharged (polar or hydrophilic) chlorpromazine-labeled amino acids located within the transmembrane domain and comprising a Ser ring framed by a Leu and Thr ring, and negatively charged amino acids bordering the former ones, constituting cytoplasmic, intermediate and outer anionic rings in an N- to C-terminal direction on M2 (Fig. I). mutations of these anionic amino acids caused changes in channel conductance, yet ““1% in the absence of divalent cations 2M. In particular, mutations in the int~ediate ring have been shown to decrease the conductance ratios of Cs+ to K” and of Rb+ to K+, and thus to alter ion selectivity, without signifi-

meproadifen mustard

cantly changing that for the physiological ions Na+ and K+ (Ref. 34). The interest then shifted towards uncharged rings. ln agreement with previous mouse muscle receptor data33*35 substitution of residues from the Thr ring in rat receptor% or Torpedo receptora increases or decreases channel conductance depending on the size of the amino acid sidechain at this position. The authors concluded that, together with the anionic intermediate ring, the Thr ring forms a short constriction within the channe13’. ln addition, for a given size, the conductance appears slightly but consistently higher with a polar instead of a hydrophobic sidechain3’. This suggests a possible ‘catalytic‘ role for the polar rings in the translocation of ions through the membrane38.

TiPS - August 1992 [Vol. 131 Only charged or polar amino acids had, until recently, been exchanged by site-directed mutagenesis. Then, mutation of the hydrophobic chlorpromazinelabelled Leu ring led to rather unexpected resulh?. Exchanging Leu247 in chick brain ar7 receptor for a polar residue (e.g. Thr) abolished inhibition by the channel blocker QX222, decreased the rate of desensitization of the response and increased the apparent affinity for acetylcholine. Such a pleiotropic effect of a point mutation might be thought of u priori as resulting from some kind of ‘folding mutation’ that would perturb the whole threedimensional architecture of the molecule. But the mutated molecuJe still possesses a functional channel and exhibits the same positive cooperativity as the wildtype receptor. More plausible interpretation thus places emphasis on the known aJlosteric properties of the acetylcholine receptor to undergo transitions between a limited number of conformations with different binding and channel properties38. For instance, equilibration with an agonist results in the stabilization of a closed ‘desensitized’ state that exhibits high affinity for agonists and for some competitive antagonists4-. If it is assumed that this (or one of these) desensitized state(s) has its channel open as a consequence of the mutation, then desensitization would become less effective. Instead, an open desensitized state would be stabilized at low agonist concentration. Two additional observations support this interpretation. First, single-channel recordings in the L247T mutant reveal a new conducting state of 8OpS (in addition to the 46pS of the wild type), which is activated by low acetylcholine concentrations39. Secondly, antagonists of the wild-type receptor, such as dihydro-fl-erythroidine, hexamethonium and (+)tubocurarine, behave as agonists when applied to the L247T mutant and activate this novel SOPS thus channel&. The mutation renders accessible to electrophysiological techniques states that are closed in the wild type. Conversely, in the wild type, this Leu ring would contribute to the closing of the ion channel in the

301

desensitized conformation of the receptor. The extension of this interpretation to the various glutamate receptors offers a plausible explanation for their rather singular propertie@. Indeed, several of the glutamate receptors display multiple channel states that are activated by a diverse selection of agonists (e.g. AMPA, kainate, quisqualate, etc.), accompanied or not by desensitization and, exceptionally, with slow rates of activation. Simple genetic events that differentially affect channel properties in the different states of a common conformational equilibrium might then account for such diversity of properties. q

•J

0

Interpretation of point-mutation effects on the functional properties of ligand-gated ion channels should no longer be restricted to local modifications within a static protein organization. A lesson to be learned is that, in contrast to the popular ‘clone-and-patch procedure, the protein should no longer be put into a ‘black box’. It might be useful to take into consideration the alJosteric properties of these receptor-gated ion channels, in particular the dynamics of their interconversion between multiple conformational states. JEAN-PIERRE ANNE

DEVILLERS-THtiRY, GAL21

AND DANIEL

CHANGEUX, JEAN-LUC BERTRAND*

Institut Pasteur, Deppartement des Biotechnologies, Neuyobiologie Molc%zulaiye,25 yue du DY Roux. 75015 Paris, France, and ‘Departement de Physiologie. Centye Medical Universitaiye, Faculte’ de Mddecine, CH-1211 Get&e 4, Switzerland.

References 1 Numa, S. (1989)Harvey Lect.83,121~165 2 Galzi, J-L., Revah, F., Bessis, A. and 3 4 5 6 7 8 9 10 11 12

Changeux, J-P. (1991) Annu. Rev. Pharmacol. Toxicol. 31,37-72 H. (1990) Biochemishy 29, Beta, 3591-3599 Cooper, E., Couturier, S. and Ballivet, M. (1991) Nature 350.235-238 Chavez;R. A. and Hall, Z. W. (1991) I. Biol. Chem. 266,15532-15538 -Yu, X. M. and Hall, Z. W. (1991) Nature 352,64-67 Chavez, R. A. and Hall, Z. W. (1992) J. Cell Biol. 116, 385-393 Gu. Y., &nacho, P., Gardner, P. and Ha& Zl W. (1991) Neuron 6,879-887 VerraR, S. and Hall, Z. W. (1992) Cell 68, 23-31 Galzi, J-L. et ~1. (1991) Proc. N~tl Acad. Sci. USA 88,5051-5055 Karlin, A. (1991) Haruey Lect. 85,71-107 Pedersen, S. E. and Cohen, J. B. (1990)

PYOC.Nat1 Acad. Sci. USA 87,W85-2789 13 Kao, P. et al. (1984) J. Fig!. Chem. 259, 11662-11665 14 Kao, P. N. and Karlin, A. (1986) 1. Biol, Chem. 261,80858088 15 Dermis, M. et al. (1988) Biochemishy 27, 2346-2357 16 Abramson, S. N., Li, Y., Culver, P. and Taylor, P. (1989) L Biol. Chem. 264, l2666-12672 . 17 Middleton, R. E. and Cohen,J. B. (1991) Biochemistry 3tL6987-6997 18 Gatzi, J-L. et al. (1990) J. Biol. Chem. 265, 10430-10437 19 Cohen, J. B., Sharp, S. D. and Mu, W. S. (1991) J. Biol. Chem. 266,23354-23364 20 h4ishina. M. et al. (1985) Nature 3l3, 364-368 21 Tomaselti, G. F., McLaughlin, J. T., Jurman, M., Hawrot, E. and YeBen, G. (1991) Biophysical I. 60.721-727 22 Gabi, J-L. et nl. (1991) FEBS L&t. 294, 198202 23 Sussman, J, et al. (1991) Scicxce 253, 872-879 24 Dougherty, D. A. and Stauffer, D. A. (1990) Science 250,1558-1560 25 O’Leary, M. E. and White, M. M. (1991) Sot. Neurosci. Absh. 17,145 26 Giraudat. 1.. Dennis. M.. Heidmann. T.. Chang, J. k: and &n&x, J-P. (1986) Proc. Nat1 Acad. Sci. USA 83.27X9-2723 27 Giraudat, J. et ~1. (1987) Bioc&misfy 26, 2410-2418 28 Oberth*k, W. et al. (1986) EMBO J= 5, 1815-1819 29 Hucho, F. L., Oberthiir, W. and Loftspeich, F. (1986) FEBS Left. 285,137-142 30 Revah, F., GaIzi, J-L., Giraudat, J., Haumont, P. Y.. Ledemr, F. and Change&, J-P. (i990) PYOC..Nat1 Acad. Sci. USA 87,4675-4679 31 Imoto. K. et ~1. (1986) NQ~u~I?324, 67&674 32 hnoto, K. et al. (1988) Natare 335, 645-648 33 Leonard, R. J., Labarca, C. G., Chamet, P., Davidson, N. and Lester, H. k (1988) Science 242,1578-1581 34 KOMO, T. et ~1. (1991) PYOC.R Sot. Lend. Ser. B 244.69-79 35 Chamet, fi. et al. (1990) Neuron 2,87-95 36 Villarroel, A., Herke, S., Koenen, Mand Sakmann, B. (1991) Pmt. R Sot. Lond. Ser. B 243,69-74 37 Imoto, K. et al. (1991) FEBS tiff. 289, 193-200 38 Changeux, J-P. (1990) Fidia Research Faundation Neuroscience Award Lectures (Vol. 4) (Changeux, J-P., LBnas, R. R., Purves, D. and Bloom, F. E., eds), pp. 21-168, Raven Press 39 Revah. F. et al. (1991) Nature 353. 8467849 40 Grtinhagen, H. H. and Change% J-P. (1976) 1. Mol. Biol. 106.497-516 41 keul&, R. R., Boyd, N. D. and Cohen, J. B. (1982) Biochemistyy 21,~3467 42 Heidmann, T., Oswald, R. E. and Changeux, J-P. (1983) BiochemistTy 22, 3112-3127 43 Bertrand, D. et ~1. (1992) Proc. NQEAcad. Sci. USA 89,1261-1265 44 Pedersen, S. E. and Cohen, J. B. (1990) Biophys. J. 57, 126s DDR n-N,N-(dimethvlamino)benxene diazotkum fluorobok MBTA: 4-(N-maleimido)benzyltrimethyl ammonium iodide Qx222: N’-(trimethylaminomethyl)-2’,6’Glidide